Comparative Transcriptome Analysis Reveals the Protective Role of Melatonin during Salt Stress by Regulating the Photosynthesis and Ascorbic Acid Metabolism Pathways in Brassica campestris.

Salinity stress is a type of abiotic stress which negatively affects the signaling pathways and cellular compartments of plants. Melatonin (MT) has been found to be a bioactive compound that can mitigate these adverse effects, which makes it necessary to understand the function of MT and its role in salt stress. During this study, plants were treated exogenously with 100 µM of MT for 7 days and subjected to 200 mM of salt stress, and samples were collected after 1 and 7 days for different indicators and transcriptome analysis. The results showed that salt reduced chlorophyll contents and damaged the chloroplast structure, which was confirmed by the downregulation of key genes involved in the photosynthesis pathway after transcriptome analysis and qRT-PCR confirmation. Meanwhile, MT increased the chlorophyll contents, reduced the electrolyte leakage, and protected the chloroplast structure during salt stress by upregulating several photosynthesis pathway genes. MT also decreased the H2O2 level and increased the ascorbic acid contents and APX activity by upregulating genes involved in the ascorbic acid pathway during salt stress, as confirmed by the transcriptome and qRT-PCR analyses. Transcriptome profiling also showed that 321 and 441 DEGs were expressed after 1 and 7 days of treatment, respectively. The KEGG enrichment analysis showed that 76 DEGs were involved in the photosynthesis pathway, while 35 DEGs were involved in the ascorbic acid metabolism pathway, respectively. These results suggest that the exogenous application of MT in plants provides important insight into understanding MT-induced stress-responsive mechanisms and protecting Brassica campestris against salt stress by regulating the photosynthesis and ascorbic acid pathway genes.

Transcriptome Analysis Reveals the Mechanism of Exogenous Selenium in Alleviating Cadmium Stress in Purple Flowering Stalks (Brassica campestris var. purpuraria).

In China, cadmium (Cd) stress has a significant role in limiting the development and productivity of purple flowering stalks (Brassica campestris var. purpuraria). Exogenous selenium supplementation has been demonstrated in earlier research to mitigate the effects of Cd stress in a range of plant species; nevertheless, the physiological and molecular processes by which exogenous selenium increases vegetable shoots' resistance to Cd stress remain unclear. Purple flowering stalks (Brassica campestris var. purpuraria) were chosen as the study subject to examine the effects of treatment with sodium selenite (Na2SeO3) on the physiology and transcriptome alterations of cadmium stress. Purple flowering stalk leaves treated with exogenous selenium had higher glutathione content, photosynthetic capacity, and antioxidant enzyme activities compared to the leaves treated with Cd stress alone. Conversely, the contents of proline, soluble proteins, soluble sugars, malondialdehyde, and intercellular CO2 concentration tended to decrease. Transcriptome analysis revealed that 2643 differentially expressed genes (DEGs) were implicated in the response of exogenous selenium treatment to Cd stress. The metabolic pathways associated with flavonoid production, carotenoid synthesis, glutathione metabolism, and glucosinolate biosynthesis were among those enriched in these differentially expressed genes. Furthermore, we discovered DEGs connected to the production route of glucosinolates. This work sheds fresh light on how purple flowering stalks' tolerance to cadmium stress is improved by exogenous selenium.

The regulatory metabolic networks of the Brassica campestris L. hairy roots in response to cadmium stress revealed from proteome studies combined with a transcriptome analysis.

Brassica campestris L., a cadmium (Cd) hyperaccumulating herbaceous plant, is considered as a promising candidate for the bioremediation of Cd pollution. However, the molecular mechanisms regulating these processes remain unclear. The present work, using proteome studies combined with a transcriptome analysis, was carried out to reveal the response mechanisms of the hairy roots of Brassica campestris L. under Cd stress. Significant tissue necrosis and cellular damage occurred, and Cd accumulation was observed in the cell walls and vacuoles of the hairy roots. Through quantitative proteomic profiling, a total of 1424 differentially expressed proteins (DEPs) were identified, and are known to be enriched in processes including phenylalanine metabolism, plant hormone signal transduction, cysteine and methionine metabolism, protein export, isoquinoline alkaloid biosynthesis and flavone biosynthesis. Further studies combined with a transcriptome analysis found that 118 differentially expressed genes (DEGs) and their corresponding proteins were simultaneously up- or downregulated. Further Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis of the 118 shared DEGs and DEPs indicated their involvement in calcium, ROS and hormone signaling-mediated response, including regulation of carbohydrate and energy metabolism, biosynthesis of GSH, PCs and phenylpropanoid compounds that play vital roles in the Cd tolerance of Brassica campestris L. Our findings contribute to a better understanding of the regulatory networks of Brassica campestris L. under Cd stress, as well as provide valuable information on candidate genes (e.g., BrPAL, BrTAT, Br4CL, BrCDPK, BrRBOH, BrCALM, BrABCG1/2, BrVIP, BrGCLC, BrilvE, BrGST12/13/25). These results are of particular importance to the subsequent development of promising transgenic plants that will hyperaccumulate heavy metals and efficient phytoremediation processes.

Establishment of hairy root system of transgenic IRT1 brassica campestris L. and preliminary study of its effect on cadmium enrichment.

Cadmium (Cd) is the main heavy metal pollutant in soil. The combination of genetic engineering technology and Rizobium rhizogenes mediated technology can effectively improve the enrichment efficiency of heavy metals in super accumulators and reduce soil heavy metal pollution. In this study, the transgenic hairy root system containing the IRT1 gene of Cd hyperaccumulator-Brassica campestris L. was successfully constructed by the R. rhizogenes mediated method (IRT1 gene come from Arabidopsis thaliana). The hairy roots of each subculture can grow stably within 6 weeks, and IRT1 gene will not be lost within 50 subcultures., which is detected using PCR method. The results of Cd enrichment experiments showed that after treatment with 100 µmol/L Cd for 14 days, the growth state of transgenic IRT1 hairy roots only showed slight browning. Also, the accumulation value of Cd reached 331.61 µg/g and the enrichment efficiency of transgenic IRT1 hairy roots was 13.8% higher than that of wild-type hairy roots. Western blotting results showed that the expression of IRT1 protein in transgenic hairy roots was significantly higher than that of wild-type hairy roots under Cd stress. The above results indicated that the overexpression of IRT1 gene can help B. campestris L. hairy roots to effectively cope with Cd stress and improve its ability to enrich Cd.

In this study, the transgenic hairy root system containing the IRT1 gene of Cd hyperaccumulator-Brassica campestris L. was successfully constructed by the Rizobium rhizogenes mediated method. At the same time, the growth state and cadmium enrichment efficiency of transgenic hairy roots under different concentrations of Cd stress were studied. Overexpression of IRT1 gene can effectively improve the tolerance of hairy root to Cd. The enrichment efficiency of transgenic IRT1 hairy roots was 13.8% higher than that of wild-type hairy roots. The transgenic IRT1 hairy root system established in this study can be used as a reliable experimental model for the study of Cd adsorption mechanism, and can be further regenerated to obtain transgenic IRT1 B. campestris L. plants for the study of heavy metal Cd pollution remediation.

Combined Metabolome and Transcriptome Analysis Elucidates Sugar Accumulation in Wucai (Brassica campestris L.).

Wucai (Brassica campestris L.) is a leafy vegetable that originated in China, its soluble sugars accumulate significantly to improve taste quality during maturation, and it is widely accepted by consumers. In this study, we investigated the soluble sugar content at different developmental stages. Two periods including 34 days after planting (DAP) and 46 DAP, which represent the period prior to and after sugar accumulation, respectively, were selected for metabolomic and transcriptomic profiling. Differentially accumulated metabolites (DAMs) were mainly enriched in the pentose phosphate pathway, galactose metabolism, glycolysis/gluconeogenesis, starch and sucrose metabolism, and fructose and mannose metabolism. By orthogonal projection to latent structures-discriminant s-plot (OPLS-DA S-plot) and MetaboAnalyst analyses, D-galactose and ß-D-glucose were identified as the major components of sugar accumulation in wucai. Combined with the transcriptome, the pathway of sugar accumulation and the interact network between 26 DEGs and the two sugars were mapped. CWINV4, CEL1, BGLU16, and BraA03g023380.3C had positive correlations with the accumulation of sugar accumulation in wucai. The lower expression of BraA06g003260.3C, BraA08g002960.3C, BraA05g019040.3C, and BraA05g027230.3C promoted sugar accumulation during the ripening of wucai. These findings provide insights into the mechanisms underlying sugar accumulation during commodity maturity, providing a basis for the breeding of sugar-rich wucai cultivars.

Heterologous Expression of Two Brassica campestris CCCH Zinc-Finger Proteins in Arabidopsis Induces Cytoplasmic Foci and Causes Pollen Abortion.

The membrane-less organelles in cytoplasm that are presented as cytoplasmic foci were successively identified. Although multiple CCCH zinc-finger proteins have been found to be localized in cytoplasmic foci, the relationship between their specific localization and functions still needs further clarification. Here, we report that the heterologous expression of two Brassica campestris CCCH zinc-finger protein genes (BcMF30a and BcMF30c) in Arabidopsis thaliana can affect microgametogenesis by involving the formation of cytoplasmic foci. By monitoring the distribution of proteins and observing pollen phenotypes, we found that, when these two proteins were moderately expressed in pollen, they were mainly dispersed in the cytoplasm, and the pollen developed normally. However, high expression induced the assembly of cytoplasmic foci, leading to pollen abortion. These findings suggested that the continuous formation of BcMF30a/BcMF30c-associated cytoplasmic foci due to high expression was the inducement of male sterility. A co-localization analysis further showed that these two proteins can be recruited into two well-studied cytoplasmic foci, processing bodies (PBs), and stress granules (SGs), which were confirmed to function in mRNA metabolism. Together, our data suggested that BcMF30a and BcMF30c play component roles in the assembly of pollen cytoplasmic foci. Combined with our previous study on the homologous gene of BcMF30a/c in Arabidopsis, we concluded that the function of these homologous genes is conserved and that cytoplasmic foci containing BcMF30a/c may participate in the regulation of gene expression in pollen by regulating mRNA metabolism.

CaFe-layered double hydroxide corn straw biochar reduced heavy metal uptake by Brassica campestris L. and Ipomoea aquatic F.: Rhizosphere effects and oxidative stress alleviation.

In the present study, CaFe-layered double hydroxide corn straw biochar (CaFe-LDH@CSB) was applied to the rhizosphere soil of both pakchoi (Brassica campestris L. ssp. Chinensis Makino, B. campestris L.) and water spinach (Ipomoea aquatic F., I. aquatic F.) to explore and clarify the potential mechanism by which CaFe-LDH@CSB helps vegetables reduce heavy metal (HM) uptake and alleviate oxidative stress. Pot experiments were conducted with CaFe-LDH@CSB applied at four levels: control (CK), T1 (5 g kg-1), T2 (10 g kg-1) and T3 (20 g kg-1). The results indicated that the application of CaFe-LDH@CSB significantly increased pH and decreased the acid-soluble forms of Cd, Pb, Zn and Cu in the rhizosphere soil of both B. campestris L. and I. aquatic F.; decreases of 39.4%, 18.0%, 10.0% and 33.3% in B. campestris L. and of 26.6%, 49.1%, 13.2% and 36.8% in I. aquatic F., respectively, were observed at the T3 level. Moreover, CaFe-LDH@CSB application reduced HM uptake by B. campestris L. and decreased HM-induced oxidative stress through the regulation of soil physicochemical properties and microbial abundance. For B. campestris L., variations in Sordariomycetes helped alleviate the accumulation of HMs in the aerial part, while GSH and -SH from the nonenzymatic system played an important role in scavenging H2O2 in leaves, thus helping B. campestris L. alleviate HM-induced oxidative stress. For I. aquatica F., variations in Vicinamibacteria and Mortierellomycetes helped alleviate the accumulation of HMs in plants, while GSH and PCs from nonenzymatic systems played an important role in removing ·O2- in leaves, thereby helping I. aquatica F. alleviate HM-induced oxidation stress. Our study indicated that the application of CaFe-LDH@CSB improved the rhizosphere soil environment and rebuilt the soil microbial community, helping B. campestris L. and I. aquatica F. alleviate HM-induced oxidative stress and promoting the growth of both vegetables.

4-phenylpyridine suppresses UVB-induced skin inflammation by targeting c-Src in vitro and in vivo.

Acute or repetitive exposure to ultraviolet (UV) cause disruptions to the skin barrier and subsequent inflammatory skin disease. 4-phenylpyridine (4-PP) is a constituent of Brassica campestris L. ssp. Pekinensis and its effect on skin inflammation and molecular target remain unclear. The purpose of this study is to confirm the anti-inflammatory efficacy of 4-PP on UVB-induced skin inflammation in human keratinocytes HaCaT and mouse skin and validation of its molecular target. 4-PP also attenuated UVB-induced phosphorylation of p38/mitogen-activated protein kinase kinase (MKK) 3/6, c-Jun N-terminal kinase 1/2, MKK 4/7, extracellular-signal-regulated kinase 1/2, mitogen-activated protein kinase 1/2. Additionally, 4-PP inhibited UVB-induced phosphorylation of epidermal growth factor receptor (EGFR) Y1068, Y1045 and 854 residues but not the proto-oncogene tyrosine-protein kinase c-Src. Drug affinity responsive target stability assay revealed that 4-PP directly binds to c-Src and inhibits pronase c-proteolysis. Knockdown of c-Src inhibited UVB-induced COX-2 expression and phosphorylation of MAPKs and EGFR in HaCaT cells. Dorsal treatment of 4-PP prevented UVB (0.5 J/cm2 )-induced skin thickness, phosphorylation of EGFR and COX-2 expression in mouse skin. Our findings suggest that 4-PP can be used as anti-inflammatory agent with an effect of skin inflammation by inhibiting the COX-2 expression via suppressing the c-Src/EGFR/MAPKs signalling pathway.

Genome-wide analysis of the DREB family genes and functional identification of the involvement of BrDREB2B in abiotic stress in wucai (Brassica campestris L.).

Dehydration responsive element binding protein (DREB) is a significant transcription factor class known to be implicated in abiotic stresses. In this study, we systematically conducted a genome-wide identification and expression analysis of the DREB gene family, including gene structures, evolutionary relationships, chromosome distribution, conserved domains, and expression patterns. A total of 65 DREB family gene members were identified in Chinese cabbage (Brassica rapa L.) and were classified into five subgroups based on phylogenetic analysis. Through analysis of the conserved domains of BrDREB family genes, only one exon existed in the gene structure. Through the analysis of cis-acting elements, these genes were mainly involved in hormone regulation and adversity stress. In order to identify the function of BrDREB2B, overexpressed transgenic Arabidopsis was constructed. After different stress treatments, the germination rate, root growth, survival rate, and various plant physiological indicators were measured. The results showed that transgenic Arabidopsis thaliana plants overexpressing BrDREB2B exhibited enhanced tolerance to salt, heat and drought stresses. Taken together, our results are the first to report the BrDREB2B gene response to drought and heat stresses in Chinese cabbage and provide a basis for further studies to determine the function of BrDREBs in response to abiotic stresses.

Transcriptome analysis and differential gene expression profiling of wucai (Brassica campestris L.) in response to cold stress.

BACKGROUND: Wucai suffers from low temperature during the growth period, resulting in a decline in yield and poor quality. But the molecular mechanisms of cold tolerance in wucai are still unclear. RESULTS: According to the phenotypes and physiological indexes, we screened out the cold-tolerant genotype "W18" (named CT) and cold-sensitive genotype "Sw-1" (named CS) in six wucai genotypes. We performed transcriptomic analysis using seedling leaves after 24 h of cold treatment. A total of 3536 and 3887 differentially expressed genes (DEGs) were identified between the low temperature (LT) and control (NT) comparative transcriptome in CT and CS, respectively, with 1690 DEGs specific to CT. The gene ontology (GO) analysis showed that the response to cadmium ion (GO:0,046,686), response to jasmonic acid (GO:0,009,753), and response to wounding (GO:0,009,611) were enriched in CT (LT vs NT). The DEGs were enriched in starch and sucrose metabolism and glutathione metabolism in both groups, and α-linolenic acid metabolism was enriched only in CT (LT vs NT). DEGs in these processes, including glutathione S-transferases (GSTs), 13S lipoxygenase (LOX), and jasmonate ZIM-domain (JAZ), as well as transcription factors (TFs), such as the ethylene-responsive transcription factor 53 (ERF53), basic helix-loop-helix 92 (bHLH92), WRKY53, and WRKY54.We hypothesize that these genes play important roles in the response to cold stress in this species. CONCLUSIONS: Our data for wucai is consistent with previous studies that suggest starch and sucrose metabolism increased the content of osmotic substances, and the glutathione metabolism pathway enhance the active oxygen scavenging. These two pathways may participated in response to cold stress. In addition, the activation of α-linolenic acid metabolism may promote the synthesis of methyl jasmonate (MeJA), which might also play a role in the cold tolerance of wucai.

Benzoic Acid, Chlorine Dioxide, and 1-Methylcyclopropene Induce Flavonoid Metabolic Shifts in Postharvest Flowering Chinese Cabbage Revealed by High-Dimensional Analytical Data.

Flowering Chinese cabbage (Brassica campestris L. ssp. chinensis var. utilis Tsen et Lee) is one of the most popular vegetables in China. However, the loss of the functional ingredients in postharvest flowering Chinese cabbage during storage is still serious, owing to the unclear causes of the metabolic shifts. Herein, benzoic acid, chlorine dioxide, and 1-methylcyclopropene (1-MCP) could maintain the quality of postharvest flowering Chinese cabbage, and 1-MCP showed the best effect. Furthermore, transcript-metabolite profiling of the treatments revealed a transcript-metabolite correlation network of the flavonoid biosynthesis pathways with a range of 3 to 3662 differentially expressed genes (DEGs) and a range of 23 to 37 differentially accumulated metabolites (DAMs). Surprisingly, 1-MCP had the best effect on shelf life among the treatments, although chlorine dioxide could stimulate the expression of four critical differential genes (Bra007142, Bra008792, Bra009358, and Bra027457) involved in delaying flavonoid degradation (hesperetin, chalcone, rutin, baicalein). As a result, our findings will help to improve our understanding of the regulation of flavonoid production in relation to the quality of postharvest flowering Chinese cabbage during storage.

Characterization and transcriptomic analysis of a novel yellow-green leaf wucai (Brassica campestris L.) germplasm.

BACKGROUND: Leaf color mutants are the ideal materials to explore the pathways of chlorophyll (Chl) metabolism, chloroplast development, and photosynthesis system. In this study, a spontaneous yellow-green leaf wucai (Brassica campestris L.) mutant "WY16-13" was identified, which exhibited yellow-green leaf color during its entire growth period. However, current understanding of the molecular mechanism underlying Chl metabolism and chloroplast development of "WY16-13" is limited. RESULTS: Total Chl and carotenoid content in WY16-13 was reduced by 60.92 and 58.82%, respectively, as compared with its wild type parental line W16-13. Electron microscopic investigation revealed fewer chloroplasts per cell and looser stroma lamellae in WY16-13 than in W16-13. A comparative transcriptome profiling was performed using leaves from the yellow-green leaf type (WY16-13) and normal green-leaf type (W16-13). A total of 54.12 million (M) (WY16-13) and 56.17 M (W16-13) reads were generated. A total of 40,578 genes were identified from the mapped libraries. We identified 3882 differentially expressed genes (DEGs) in WY16-13 compared with W16-13 (i.e., 1603 upregulated genes and 2279 downregulated genes). According to the Gene Ontology (GO) term and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses, these DEGs are involved in porphyrin and Chl metabolism [i.e., chlorophyllase (CLH), heme oxygenase (HO), chlorophyll (ide) b reductase (NYC), and protochlorophyllide oxidoreductase (POR) genes], carbohydrate metabolism, photosynthesis, and carbon fixation in photosynthetic organisms. Moreover, deficiency in Chl biosynthetic intermediates in WY16-13 revealed that the formation of the yellow-green phenotype was related to the disorder of heme metabolism. CONCLUSIONS: Our results provide valuable insights into Chl deficiency in the yellow-green leaf mutant and a bioinformatics resource for further functional identification of key allelic genes responsible for differences in Chl content.

Exogenous inoculation of endophytic bacterium Bacillus cereus suppresses clubroot (Plasmodiophora brassicae) occurrence in pak choi (Brassica campestris sp. chinensis L.).

MAIN CONCLUSION: The presence of Bacillus cereus plays a key role in clubroot suppression and improves plant biomass in pak choi. B. cereus is reported for the first time as a novel biocontrol agent against clubroot. Plasmodiophora brassicae Woronin causes a devastating infectious disease known as clubroot that is damaging to cruciferous vegetables. This study aimed to isolate beneficial bacteria from the rhizosphere soil of pak choi (Brassica campestris sp. chinensis) and to evaluate the ability of the isolate to reduce the severity of clubroot. Strains obtained from the rhizosphere of symptomless pak choi were first selected on the basis of their germination inhibition rate and effects on the viability of P. brassicae resting spores. Eight bacterial isolates had inhibitory effects against the resting spores of clubroot causing pathogen. However, MZ-12 showed the highest inhibitory effect at 73.4%. Inoculation with MZ-12 enhanced the plant biomass relative to plants grown without MZ-12 as well as P. brassicae infected plants. Furthermore, enhanced antioxidant enzymatic activities were observed in clubroot-infected plants during bacterial association. Co-inoculation of the plant with both P. brassicae and MZ-12 resulted in a 64% reduction of gall formation in comparison to plants inoculated with P. brassicae only. Three applications of MZ-12 to plants infected with P. brassicae at 7, 14 and 21 days after seeding (DAS) were more effective than one application and repressed root hair infection. According to 16S rDNA sequence analysis, strain MZ-12 was identified as had a 100% sequence similarity with type strain Bacillus cereus. The findings of the present study will facilitate further investigation into biological mechanisms of cruciferous clubroot control.

Nitrogen and phosphorus concentrations in growth media affect the relationship between root endophytic fungi and host plant.

Endophytic fungi (EPF) colonize plant roots and enhance their growth. The relationship between host plant and EPF can be affected by several factors, such as growth media, host species, and fungal species. The objective of this study was to clarify the effect of nutrient concentration in growth media on the relationship between host plant and root EPF. Brassica campestris was grown in 1/100 Murashige and Skoog (MS), 1/10 MS, 1/100 MS and 1/10 nitrogen (high N), and 1/100 MS and 1/10 phosphorus (high P) media. B. campestris was inoculated with four root EPFs isolated from forest soils in Indonesia and harvested 28 days after transplant. Shoot dry weight (SDW) and colonization in roots were measured. All the isolates colonized roots of B. campestris. Two isolates increased the SDW of B. campestris grown on 1/100 MS media. The shoot growth response of B. campestris to EPF colonization on 1/100 MS was higher than that on 1/100 high N and 1/100 high P MS media. These results suggest that concentration of nitrogen and phosphorus in growth media determine the relationship between B. campestris and root EPF.

Cabbage cultivars influence transfer and toxicity of cadmium in soil-Chinese flowering cabbage Brassica campestris-cutworm Spodoptera litura larvae.

We executed a pot experiment to examine the differences of absorption, chemical forms, subcellular distribution, and toxicity of Cd between two cultivars of Chinese flowering cabbage Brassica campestris [Lvbao701 (low-Cd cultivar) and Chicaixin No.4 (high-Cd cultivar)]. Compared to Chicaixin No.4, the presence of Lvbao701 enhanced the proportion of insoluble Cd forms in soil, Lvbao701 roots and leaves had higher proportion of Cd converted into insoluble phosphate precipitates and pectate-or protein-bound forms and lower proportion of inorganic Cd, which result in low accumulation and toxicity of Cd to Lvbao701 and cutworm Spodoptera litura fed on Lvbao701 leaves. Instead of total Cd, Cd transfer and toxicity in B. campestris-S. litura system depend on chemical Cd forms in soil and cabbages and subcellular Cd distributions in cabbages and insects, and the proportions of them were not the highest among all chemical forms and subcellular distributions of Cd. Although exchangeable Cd was major Cd chemical form in cabbage planted soil, Cd bound to iron and manganese oxides and to organic matter were significantly correlated with growth indices and photosynthesis parameters of cabbages. Despite major part of Cd was precipitated in cell wall of roots, Cd in organelle fraction was closely associated with the fitness of cabbages. Metal-rich granules, not cytosolic fraction (the major subcellular Cd distribution), affected the food utilization of S. litura. Therefore, cabbage cultivars significantly affected Cd transfer and toxicity in B. campestris-S. litura system, and the use of Lvbao701 in Cd polluted soil could reduce potential risks for Cd entering food chains.

Relationships between soil properties and the accumulation of heavy metals in different Brassica campestris L. growth stages in a Karst mountainous area.

The speciation and activity of heavy metals in farmland were changed with the different soil properties and flooded environment, especially in the complex and rainy environment in soil of Guizhou Province. The objective of this study was to explore the concentrations of a variety of heavy metal activity and the speciation of those heavy metals in rhizosphere soil at different growth stages of Brassica campestris L. in a Karst mountainous area. Tessier's five-stage sequential extraction procedure, the potential ecological risk index, a Bayesian network, accumulation factors, translocation factors and a laboratory simulation experiment were applied in this study. The results showed that (1) no heavy metal concentrations (except the Cd concentration) exceeded the limits of the soil environmental quality risk control standards for soil contamination of agricultural land in China (GB15618-2018). (2) The orders of the accumulation factor and translocation factor values were Zn > Cd > Cu > Pb > Cr and Cd > Cu > Zn > Pb > Cr, respectively. The order of the heavy metal contents of different tissues during the whole growth period was roots > leaves > stems. (3) The indoor simulation test exhibited that the dry-wet alternation and flooding can reduce Cd activity in soil. (4) Redox potential (Eh), rather than pH or organic matter, was the main factor impacting the total content and chemical speciation of heavy metals in the soil, based on a dynamic Bayesian network. Based on the results, we suggest that the activity of heavy metals should be improved by using dry-wet alternation, whereas the proportions of ion-exchangeable forms of heavy metals are relatively low in the study area (except for Cd). Several measures may be taken to enhance soil acidity and reduce the Cd activity during Brassica campestris L. cultivation.

BcMF30a and BcMF30c, Two Novel Non-Tandem CCCH Zinc-Finger Proteins, Function in Pollen Development and Pollen Germination in Brassica campestris ssp. chinensis.

Chinese cabbage (Brassica campestris) is an economically important leaf vegetable crop worldwide. Mounting studies have shown that cysteine-cysteine-cysteine-histidine (CCCH) zinc-finger protein genes are involved in various plant growth and development processes. However, research on the involvement of these genes in male reproductive development is still in its infancy. Here, we identified 11 male fertility-related CCCH genes in Chinese cabbage. Among them, a pair of paralogs encoding novel non-tandem CCCH zinc-finger proteins, Brassica campestris Male Fertility 30a (BcMF30a) and BcMF30c, were further characterized. They were highly expressed in pollen during microgametogenesis and continued to express in germinated pollen. Further analyses demonstrated that both BcMF30a and BcMF30c may play a dual role as transcription factors and RNA-binding proteins in plant cells. Functional analysis showed that partial bcmf30a bcmf30c pollen grains were aborted due to the degradation of pollen inclusion at the microgametogenesis phase, and the germination rate of viable pollen was also greatly reduced, indicating that BcMF30a and BcMF30c are required for both pollen development and pollen germination. This research provided insights into the function of CCCH proteins in regulating male reproductive development and laid a theoretical basis for hybrid breeding of Chinese cabbage.

The distinct functions of two classical arabinogalactan proteins BcMF8 and BcMF18 during pollen wall development in Brassica campestris.

Arabinogalactan proteins (AGPs) are extensively glycosylated hydroxyproline-rich glycoproteins ubiquitous in all plant tissues and cells. AtAGP6 and AtAGP11, the only two functionally known pollen-specific classical AGP encoding genes in Arabidopsis, are reported to have redundant functions in microspore development. BcMF18 and BcMF8 isolated from Brassica campestris are the orthologues of AtAGP6 and AtAGP11, respectively. In contrast to the functional redundancy of AtAGP6 and AtAGP11, single-gene disruption of BcMF8 led to deformed pollen grains with abnormal intine development and ectopic aperture formation in B. campestris. Here, we further explored the action of BcMF18 and its relationship with BcMF8. BcMF18 was specifically expressed in pollen during the late stages of microspore development. Antisense RNA transgenic lines with BcMF18 reduction resulted in aberrant pollen grains with abnormal cellulose distribution, lacking intine, cytoplasm and nuclei. Transgenic plants with repressive expression of both BcMF8 and BcMF18 showed a hybrid phenotype, expressing a mixture of the phenotypes of the single gene knockdown plant lines. In addition, we identified functional diversity between BcMF18/BcMF8 and AtAGP6/AtAGP11, mainly reflected by the specific contribution of BcMF18 and BcMF8 to pollen wall formation. These results suggest that, unlike the orthologous genes AtAGP6 and AtAGP11 in Arabidopsis, BcMF18 and BcMF8 are both integral to pollen biogenesis in B. campestris, acting through independent pathways during microspore development.

Evolution of MIR159/319 genes in Brassica campestris and their function in pollen development.

KEY MESSAGE: MIR159/319 have conserved evolution and diversified function after WGT in Brassica campestris, both of them can lead pollen vitality and germination abnormality, Bra-MIR319c also can function in flower development. MiR159 and miR319 are extensively studied highly conserved microRNAs which play roles in vegetative development, reproduction, and hormone regulation. In this study, the effects of whole-genome triplication (WGT) on the evolution of the MIR159/319 family and the functional diversification of the genes were comprehensively investigated in Brassica campestris. We identified 11 MIR159/319 genes in B. campestris, which produced five mature sequences. After analyzing the precursor sequences and phylogenetic tree, we found that Bra-MIR159/319 have evolutionary conservatism. Furthermore, Bra-MIR159/319 show functional diversification after WGT, as indicated by their expression patterns and the cis-element in their promoter. GUS signal showed that Bra-MIR159a and Bra-MIR319c can be expressed in anther but in different development stages. In B. campestris, overexpressed MIR159a and MIR319c contribute to late anther development and promote pollen abortion. Moreover, Bra-MIR319c can partially assume the function of MIR319a in flower development.

BcPME37c is involved in pollen intine formation in Brassica campestris.

Pollen wall development is one of the key processes of pollen development. Several pectin methylesterase (PME) genes participate in pollen germination and pollen tube growth. However, the relationship between PME genes and pollen intine formation remains unclear. In this study, we investigated the expression and subcellular localization of the PME gene BcPME37c in Brassica campestris. Furthermore, morphology and cytology methods were used to examine the phenotype of the CRISPR/Cas9 system-induced BcPME37c mutant. We found that BcPME37c is predominately expressed in mature stamen and located at the cell wall. BcPME37c mutation causes the abnormal thickening of the pollen intine of B. campestris. Our study indicated that BcPME37c is required for pollen intine formation in B. campestris.